Lightning Oral Talk 25th Annual Lorne Proteomics Symposium 2020

Development of a primary cell proficient phosphoproteomic workflow (#62)

Rune H Larsen 1 2 , Jarrod Sandow 1 , Laura Dagley 1 , Andrew Webb 1
  1. Walter and Eliza Hall Institute, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia

Phosphorylation is a critical post translational modification (PTM) that occurs on proteins to modulate the propagation of intracellular signals. Phosphoproteomics has established itself as the tool of choice to investigate the complexities of intracellular signalling cascades in an unbiased and highly sensitive manner. Phosphoproteomics is able to delineate how signals change when exposed to different stimuli or pathological conditions in time.


However, the utility of phosphoproteomics has been somewhat hampered by the relatively large amount of input material required to enable sufficient detection of protein phosphorylation abundance differences (~200 ug). This limitation has restricted the majority of phosphoproteomics studies to in vitroexperiments or mixed tissues whereas the true power of phosphoproteomics is likely to be revealed from studies in specific subsets of primary cells. The advent of improved protein digestion and phosphopeptide enrichment methods using optimized buffers and cleaner digests have vastly decreased the sample amounts required. Secondly, the increases in speed and sensitivity of mass spectrometers such as the TimsTOF Pro have greatly aided the identification of phosphorylated peptides without the need for extensive fractionation. The TimsTOF pro with its PASEF acquisition strategy vastly increases the sensitivity and MS/MS acquisition speed while maintaining high quality spectra.


Here we present a combined workflow that utilizes our USP3 acid hydrolysis workflow coupled to an Fe-IMAC enrichment protocol analysed on a phosphoproteomics optimized timsTOF Pro. we show the capabilities of the workflow using low sample amounts derived from purified primary cells (£20 ug).